Project Summary/Abstract Transcriptional enhancers, promoters and insulators are key gene regulatory elements that play important roles in the control of cell state and development, and variation in these elements has been implicated in many diseases. Interactions between enhancers and promoters generally occur within insulated neighborhoods, which are DNA loops that encompass enhancers and their target genes, formed by the interaction of two CTCF-bound sites together with cohesin. This structuring of the genome into insulated neighborhoods, which are components of Topologically Associating Domains, is essential for proper control of gene expression programs and cell identity; disease-associated variants often affect enhancer, promoter or insulator sites. Gaps in our knowledge of the factors that contribute to the structure the genome limit our understanding of the mechanisms by which these elements contribute to specific cell states in health and disease. We hypothesize that additional novel protein and RNA factors contribute to specific enhancer-promoter interactions and insulator element interactions, and have preliminary evidence supporting this concept. The goals of this proposal are to identify and characterize novel factors that structure enhancers, promoters and insulators, to decipher how they contribute to global gene control, and to determine whether disease-associated variants in enhancer, promoter and insulator elements impact gene control through these novel components. To accomplish these goals, the specific aims of the proposal are: 1) Identify and characterize DNA structuring proteins that contribute to enhancer-promoter interactions and insulator-element interactions, 2) Investigate the potential role of RNA in enhancer- promoter interactions and in insulator element interactions, and 3) Identify genomic variants that alter binding of structuring factors to enhancers, promoters and insulators. Improved understanding of the regulatory factors that structure these gene regulatory elements should continue to produce new insights into the control of cell state, reveal the key themes that operate to control gene expression programs in mammalian cell types, and produce new insights into disease mechanisms.